| Background and ObjectivesNasopharyngeal carcinoma (NPC) is one of the most common cancers in Southern China. Current treatment strategies focus on radiotherapy and chemotherapy. Early-stage NPC can be cured only by radiotherapy. However, as for the advanced NPC, treatments with radiotherapy, chemotherapy, and radiotherapy combined with chemotherapy, or even high dose chemotherapy with hematopoietic stem cell transplantation control the locoregional tumor, they do not prevent the appearance of distant metastases that affect the overall survival rate severely. Consequently, there is a desperate need for new effective therapeutic modalities. The combining application of radiotherapy, chemotherapy and monoclonal antibody may improve the effects. Combining biotherapy with conventional treatment will lead to new directions of research.In allogeneic hematopoietic transplantation, donor-derived allo-reactive natural killer (Allo-NK) cells have shown improved cure rate in hematopoietic malignancies. Allo-NK cells have recently been shown to kill melanoma, renal carcinoma, intestinal carcinoma, and ovarian cancer cells in vitro. Nevertheless, it is unknown whether Allo-NK cells have an effect on NPC cells.NK cells are important effectors of the innate immune system with a critical role in early host defense against invading pathogens and transformed cells. NK cells exert their functions through pattern recognition regulated by their specific receptors interaction with their respective ligands. Cytotoxicity of NK cells is regulated both by HLA-Killer Immunoglobulin-like Receptors (KIRs) (HLA-KIR) inhibitory signals and NKG2D ligand-NKG2D (NKG2DL-NKG2D) activating signals. The recent study indicates that the relationship between NK cells and cancer cells is much more than killing and be killed while these two cells contact, but with the existence of immunoediting results in the changes of receptor-ligand molecules and biologic behavior in both sides. Regulating molecules related to immunoediting would change the interreaction between Allo-NK cells and tumor cells. However, immunoediting and regulating between Allo-NK cells and various tumor cells are different.We hypothesize that Allo-NK cells contacting with NPC cells would generate immunoreaction on the basis of the origin, etiology, pathogenesis and biologic behavior of NPC. In this study, we investigate the antineoplastic effects of Allo-NK cells on CNE2 cells, the immunoediting between CNE2 cells and Allo-NK cells and the molecular mechanism underlying immunoediting. Furthermore, we explored the methods to exert the most cytotoxic effects on CNE2 cells via regulating the function of Allo-NK cells and increasing the sensitivity of CNE2 cells to Allo-NK cells killing. This will provide feasibility for NK-based immunotherapy in NPC. Moreover, this will provide theoretical and experimental basis for establishment of genetic-based individual adoptive immuno-chemotherapeutic regimens.MethodsPart 1 Detection of HLA-class I molecules and NKG2D ligands in CNE2 and K562 cellsDNA was extracted from CNE2 cells and K562 cells with QIAamp DNA extracting kit. The HLA genotypes of CNE2 cells were determined by sequence specific primer polymerase chain reproduction (PCR-SSP, Biotest). mRNA expressions of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, and ULBP3) were analyzed by RT-PCR. HLA-class I molecules and NKG2D ligands MICA, MICB, ULBP1, ULBP2, and ULBP3 of CNE2 and K562 cells were measured by flow cytometry(FCM).Part 2 Cytotoxicity assays of Allo-NK cells against CNE2 and K562 cellsNormal peripheral blood (PB) was obtained with informed consent. DNA was extracted from PB drawn in acid citrate dextrose buffer (ACD-B) by QIAamp DNA extracting kit. KIRs typing analysis was performed by PCR-SSP assay. NK cells were obtained from PB lymphocytes (PBLs) by CD56 antibody magnetic isolation. NK cells were cultured in the presence of recombinant human IL-2 (rhIL-2) for 24 h in RPMI 1640 complete medium. Cytotoxicity of NK cells against CNE2 and K562 cells was detected by LDH releasing assay at different effector-to-target (E: T) cell ratios. To test whether NK cells cytolytic effects were associated with NKG2D ligands and HLA-class I molecules on the surface of target cells, we blocked NKG2D ligands MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA-class I molecules by different monoclonal antibodies and assessed their killing activities at the E: T ratio of 20:1.Part 3 Immunoediting between Allo-NK cells and CNE2 or K562 cells 3.1 Immunoediting between Allo-NK cells and CNE2 cellsAllo-NK cells were isolated from 3 healthy donors as described previously. Two groups were designed into non-edited Allo-NK cells group (obtained by cultivated with 100U/ml rhIL-2), edited Allo-NK cells group (obtained by mixed with CNE2 cells at the E:T ratio of 10:1 with 100U/ml rhIL-2). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D on edited and non-edited Allo-NK cells at Oh, 4h, 24h, 48h were measured by FCM. Expressions of HLA-class I molecules and MICA/MICB, ULBP2 of non-edited and edited CNE2 cells were detected as well. Cytolytic effects of non-edited or edited Allo-NK cells against non-edited CNE2 cells and of non-edited NK cells against edited CNE2 cells were measured by LDH releasing assay.3.2 Immunoediting between Allo-NK cells and K562 cellsThe expressions of NKG2D ligands MICA/MICB on K562 cells and KIR2DL1, KIR3DL1, and NKG2D on Allo-NK cells were detected by FCM after Allo-NK cells co-cultured with K562 cells for 24 h at the E:T ratio of 1:1. The expressions of all the receptors and ligands above on non-edited Allo-NK cells and non-edited K562 cells were detected as well. The cytotoxicity of edited and non-edited NK cells was measured by LDH releasing assay.Part 4 Regulation of biologic behavior of Allo-NK cells and CNE2 cells 4.1 Effects of blocking inhibitory KIR-HLA signal pathway on cytotoxicity of Allo-NK cells against CNE2 cells(1) Effects of blocking inhibitory KIR-HLA signal pathway on cytotoxicity of non-edited Allo-NK cells against edited CNE2 cellsInhibitory KIRs of NK cells and KIR ligands HLA-class I molecules of CNE2 cells were blocked by monoclonal antibodies. Anti-tumor activity of non-edited Allo-NK cells against edited CNE2 cells at the E: T ratio of 20:1 was measured by LDH releasing assay.(2) Effects of blocking inhibitory KIR-HLA signal pathway on cytotoxicity of edited Allo-NK cells against non-edited CNE2 cellsInhibitory KIRs of Allo-NK cells and KIR ligands HLA- class I molecules of CNE2 cells were blocked by monoclonal antibodies. Cytotoxicity of edited Allo-NK cells against non-edited CNE2 cells at the E: T ratio of 20:1 was measured by LDH releasing assay. 4.2 Effects of regulating NKG2DL-NKG2D signal pathways on the cytotoxicity of Allo-NK cells against CNE2 cells(1) Effects of rhIL-2 and rhIL-15 on the expressions of NKG2D and the cytotoxicity of edited Allo-NK cells against CNE2 cellsAllo-NK cells were purified as described previously. Four groups were designed into non-edited Allo-NK cells group (obtained by cultivated with 100 U/ml rhIL-2 for 24 h), edited Allo-NK cells group (obtained by mixed the Allo-NK cells with the CNE2 cells at the ratio of 10:1 with 100U/ml rhIL-2 for 24 h), edited Allo-NK cells cultured with 1000U/ml rhIL-2 group, and edited Allo-NK cells cultivated with 10ng/ml rhIL-15 group. Expressions of NKG2D in each group were detected by FCM. Cytolytic effects of every single group of Allo-NK cells mentioned above against non-edited or edited CNE2 cells were measured by LDH releasing assay.(2) Effects of DDP on NKG2D ligands MICA/MICB, ULBP2 expressions on edited or non-edited CNE2 cells and on CNE2 cells sensitivity to Allo-NK cells cytotoxicityEdited or non-edited CNE2 cells were seeded into 24-well tissue culture plate at a cell density of 1×10~5/ml. The expressions of MICA/MICB and ULBP2 of CNE2 cells were determined by FCM after CNE2 cells were treated with various concentrations of DDP for 24 h. The differences between DDP-treated and no DDP-treated CNE2 cells in sensitivity to Allo-NK cells cytotoxicity were measured by LDH releasing assay.(3) Effects of 5-Fu on NKG2D ligands MICA/MICB, ULBP2 expressions on edited or non-edited CNE2 cells and on CNE2 cells sensitivity to NK cells cytotoxicityEdited or non-edited CNE2 cells were seeded into 24-well tissue culture plate at a cell density of 1×10~5/ml. The expressions of MICA/MICB and ULBP2 of CNE2 cells were determined by FCM after CNE2 cells were treated with control or 5μM 5-Fu for 24 h. The differences between 5-Fu-treated and no 5-Fu-treated CNE2 cells in sensitivity to Allo-NK cells cytotoxicity were measured by LDH releasing assay.Statistical analysis The calculation was performed using SPSS 13.0 software package. The data represented as the mean±standard deviation. Comparison in experiments was performed using one-way analysis of variance (ANOVA), independent-samples t-test and repeated measurement of analysis of variance toassess the statistical significance of differences between groups. Differences with P <0.05 were considered to be significant.ResultsPart 1 Expressions of KIR ligands HLA-class I molecules and NKG2D ligands on CNE2 and K562 cells1.1 HLA genotypes in CNE2 cellsHLA-A, B, Cw genotypes of CNE2 cells were A2, 24; B18, 35; Cw4, 7. These data showed that CNE2 cells are HLA-C1, C2 isotype.1.2 Expressions of NKG2D ligands on CNE2 and K562 cellsMICA, MICB, ULBP1, ULBP2, and ULBP3 were expressed on CNE2and K562 cells detected by RT-PCR.1.3 Expressions of KIR ligands HLA-class I molecules and NKG2D ligands on CNE2 and K562 cellsCNE2 cells expressed NKG2D ligands MICA, MICB, ULBP2 and HLA-class I molecules but no ULBP1 and ULBP3. However, K562 cells expressed MICA, MICB, ULBP1, ULBP2, and ULBP3 ligands but no HLA-class I molecules. The data indicated that CNE2 cells expressed two kinds of KIR-binding ligands and three kinds of NKG2D-binding ligands. However, K562 cells expressed five kinds of NKG2D-binding ligands but no ligands integrated to NK cells KIRs. Part 2 Cytotoxic effects of Allo-NK cells on CNE2 and K562 cells 2.1 KIR genotypes of Allo-NK cellsThe KIR genotypes of three healthy donors were KIR2DL1, KIR2DL3, KIR3DL1, and KIR3DL2. This showed the existence of mismatch between KIR3DL1, KIR3DL2 and HLA-A, B molecules on CNE2 cells.2.2 Cytotoxic effects of Allo-NK cells on CNE2 and K562 cellsCytotoxic effects of Allo-NK cells on CNE2 and K562 cells were enhanced in relation to the increased E: T ratios. The cytotoeixity of Allo-NK cells against CNE2 cells was significantly lower compared with that against K562 cells at various levels of E: T ratios (t = 16.136, P = 0.000; t = 15.909, P = 0.000; t = 10.622, P = 0.006; t = 10.349, P = 0.000, respectively). At the E: T ratio of 20:1, the cytotoeixity of Allo-NK cells against CNE2 cells pretreated with AMO-1, BMO-1, and M310 to block NKG2D ligands MICA, MICB, and ULBP2 were (26.59±3.85)%, (26.80±3.71)% and (26.75±3.68)% while the results in the untreated CNE2 cells were (35.74±3.59)%. Cytotoxicity was significantly decreased (P = 0.007, P = 0.008, P = 0.007) in every treated group when compared with the results in the control group. However, the cytotoeixity of NK cells against CNE2 cells pretreated with M295 or M551 to block NKG2D ligands ULBP1 and ULBP3 were (35.81±4.23)% and (36.08±3.33)%, there was no significant difference while comparing with the results in the untreated CNE2 cells (P = 0.982 and P = 0.907, respectively). The cytotoeixity of NK cells against K562 cells pretreated with AMO-1, BMO-1, M295, M310, and M551 to block NKG2D ligands MICA, MICB, ULBP1, ULBP2, and ULBP3 were (46.21±3.29)%, (49.33±0.90)%, (51.08±2.19)%, (51.04±1.62)% and (52.11±1.71)% while the results in the untreated K562 cells were (58.37±0.87)%. Cytotoxicity was significantly decreased (P = 0.000, P =0.000, P = 0.001, P =0.001, P = 0.002, respectively) in every treated group when compared with the results in the control group. Cytotoxicity of NK cells against CNE2 cells pretreated with W632 to block HLA-class I molecules was significantly increased (P = 0.000) comparing with that in the control group [(54.27±1.64)% vs (35.74±3.59)%].These findings suggested that CNE2 cells were moderate sensitive to Allo-NK cells cytolytic effects. The cytotoxicity was regulated by both HLA-KIR and NKG2DL-NKG2D signal pathways. However, cytotoxicity of Allo-NK cells against K562 cells was regulated by NKG2DL-NKG2D activating signal pathway but not inhibitory signal pathway. Thus, K562 cells were highly sensitive to Allo-NK cellscytolytic effects.Part 3 Immunoediting between Allo-NK cells and CNE2 cells or K562 cells3.1.Immunoediting between Allo-NK cells and CNE cells(1) Effects of immunoediting on the expressions of KIR and NKG2D receptors on NK cellsNK cells were cultured with or without CNE2 cells for 0 h, 4 h, 24 h, and 48 h, respectively. The expressions of KIR2DL1, KIR2DL3, and NKG2D were significantly different between Allo-NK cells cultured with and without CNE2 cells (F = 27.176, P = 0.006; F = 439.932, P = 0.000; F = 8635.791, P = 0.000, respectively). The expressions of KIR2DL1, KIR2DL3, and NKG2D were significantly different at different time points (F=12.013, P = 0.001; F= 13.799, P = 0.000; F =1145.111, P = 0.000, respectively). However, the expression of KIR3DL1 displayed no significant difference between NK cells cultured with and without CNE2 cells (F = 0.305, P = 0.610). The expression of KIR3DL1 displayed no significant difference at different time points (F = 0.185, P = 0.905). The results indicated that expressions of KIR were up-regulated and while that of NKG2D were down-regulated on NK cells cultured with CNE2 cells. (2) The effects of immunoediting on the expressions of KIR ligands and NKG2D ligands on CNE2 cellsThe expressions of HLA-class I molecules have no significant difference between non-edited and edited CNE2 cells [(99.77±0.12)% vs (97.80±0.87)%, t = 3.874, P = 0.057]. Nevertheless, the expressions of MICA/MICB were distinguished between non-edited and edited CNE2 cells [(88.67±2.81)% vs (34.53±3.15)%, t = 22.195, P = 0.000]. The expressions of ULBP2 have no significant difference between non-edited and edited CNE2 cells [(50.03±1.95)% vs (46.67±1.06)%, t = 2.627, P = 0.058]. The data showed that immunoediting induced distinguished down-expression of NKG2D ligands MICA/MICB but had no effect on HLA-class I molecules and ULBP2 on the surface of CNE2 cells.(3) Effects of immunoediting on cytotoxicity of NK cells against CNE2 cellsAt the E:T ratio of 10:1, the cytotoxicity of edited-NK cells significantlydecreased while comparing with that of non-edited NK cells [(2.74±1.64)% vs (28.08±1.79)%, t = 18.081, P = 0.000]. Similar to the results at the E:T ratio of 10:1, the cytotoxicity of edited-NK cells at the E: T ratio of 20:1 significantly decreased while comparing with that of non-edited NK cells [(4.57±2.41)% vs (36.64±3.55)%, t = 12.945, P= 0.000]. The findings suggested that immunoediting resulted in the decreases of NK cells cytotoxicity.(4) Effects of immunoediting on the sensitivity of CNE2 cells to Allo-NK cells cytolytic effectsAt the E: T ratios of 10:1 and 20:1, the cytotoxicity of Allo-NK cells against non-edited and edited CNE2 cells were (28.08±1.79)% vs (6.60±0.86)% and (36.64±3.55)% vs (11.57±2.02)%, immunoediting increased the resistance of CNE2 cells to Allo-NK cells cytolytic effects distinguishedly (t =18.741, P = 0.000 and t = 10.642, P = 0.001, respectively). 3.2 Immunoediting between Allo-NK cells and K562 cells(1) Effects of immunoediting on the expressions of KIRs and NKG2D on Allo-NK cellsThe expressions of KIR2DL1 and KIR3DL1 had no significant difference in edited Allo-NK cells comparing with non-edited Allo-NK cells [(47.20±1.08)% vs (45.70±1.22)%, t = 1.596, P = 0.186, and (35.60±1.35)% vs (34.03±1.20)%, t = 1.504, P =0.207, respectively] . However, the expressions of NKG2D decreased markedly on edited Allo-NK cells in comparison with non-edited Allo-NK cells [(34.67±3.88)% vs (98.27±0.67)%, t = 27.974, P = 0.000]. The results indicated that immunoediting induced down-expression of NKG2D activating receptors but no effects on KIRs on the surface of Allo-NK cells.(2) Effects of immunoediting on the expressions of NKG2D ligands on K562 cellsThe expressions of MICA/MICB had significant difference between non-edited and edited K562 cells [(79.90±0.87)% vs (35.56±1.01)%, t = 57.724, P= 0.000]. The data showed that immunoediting induced significant down-expression of NKG2D ligands MICA/MICB on K562 cells.(3) Effects of immunoediting on cytotoxicity of Allo-NK cells against K562 cellsThe cytotoxicity of edited Allo-NK cells decreased significantly while comparing with that of non-edited Allo-NK cells [(16.95±2.00)% vs (52.34±2.22)%, P = 0.000]. The findings suggested that immunoediting resulted in the decreases of Allo-NK cells cytotoxicity.(4) Effects of immunoediting on the sensitivity of K562 cells to Allo-NK cells cytolytic effectsThe cytotoxicity of Allo-NK cells against non-edited and edited K562 cells were (52.34±2.22)% vs (24.07±0.58)%, it showed that immunoediting significantly increased the resistance of K562 cells to NK cells cytolytic effects (P= 0.000).Part 4 Regulating biologic behavior of Allo-NK cells and CNE2 cells4.1 Effects of blocking inhibitory KIR-HLA signal pathway on the cytotoxicity of Allo-NK cells against CNE2 cells(1) Effects of blocking inhibitory KIR-HLA signal pathway on the cytotoxicity of non-edited Allo-NK cells against edited CNE2 cellsAt the E: T ratio of 20:1, the cytotoxicity of non-edited Allo-NK cells pretreated with EB6b (anti-KIR2DLl) and GL183(anti-KIR2DL2/3) against edited CNE2 cells were (19.70±1.50)% and (21.20±1.43)%, respectively. That was significant difference while comparing with that of non-edited Allo-NK cells without blockage [(12.23±1.75)%, P = 0.000, P = 0.000]. The cytotoxicity of Allo-NK cells pretreated or no pretreated with Z27 (anti- KIR3DL1) against edited CNE2 cells were no significant difference [(12.61±1.06)% vs (12.23±1.75)%, P= 0.782]. The cytotoxicity of non-edited Allo-NK cells against CNE2 cells pretreated with W632 to block HLA-class I molecules was significantly increased comparing with that of NK cells against CNE2 cells with no blockage [(37.38±2.11)% vs (12.23±1.75)%, P = 0.000]. These findings suggested blocking receptors or ligands of KIR-HLA signal pathways could enhance the cytotoxicity of non-edited Allo-NK cells.(2) Effects of blocking inhibitory KIR-HLA signal pathway on the cytotoxicity of edited Allo-NK cells against non-edited CNE2 cellsAt the E: T ratio of 20:1, the cytotoxicity of edited Allo-NK cells pretreated with EB6b (anti-KIR2DLl) and GL183 (anti-KIR2DL2/3) against non-edited CNE2 cells were (20.92±2.97)% and (19.52±0.84)%, respectively. It has significant difference while comparing with that of edited Allo-NK cells without blockage [(4.70±1.44)%, P = 0.000, P= 0.000]. The cytotoxicity of edited Allo-NK cells pretreated or no pretreated with Z27 (anti-KIR3DLl) against non-edited CNE2 cells had no significant difference [(4.94±2.36)% vs (4.70±1.44)%, P = 0.891]. The cytotoxicity of edited Allo-NK cells against non-edited CNE2 cells pretreated with W632 to block HLA-class I molecules was significantly increased comparing with that of Allo-NK cells against CNE2 cells unblocked [(33.54±2.18)% vs (4.70±1.44)%, P=0.000]. These findings suggested that blocking receptors or ligands of KIR-HLA signal pathway could improve the cytotoxicity of edited Allo-NK cells.4.2 Effects of regulating NKG2DL-NKG2D signal pathways on the cytotoxicity of Allo-NK cells against CNE2 cells(1) Effects of rhIL-2 and rhIL-15 on the expressions of NKG2D and thecytotoxicity of edited Allo-NK cells against CNE2 cellsThe expressions of NKG2D on non-edited Allo-NK cells, edited Allo-NK cells,edited Allo-NK cells cultured with rhIL-2, and edited Allo-NK cells cultured with rhIL-15 were (97.63±0.83)%, (53.50±1.25)%, (94.47±1.00)%, and (98.07±0.21)%, respectively. The differences of NKG2D expressions were significant between edited Allo-NK cells cultured with rhIL-2 or rhIL-15 and edited Allo-NK cells (P = 0.000, P = 0.000), that were also significant between edited Allo-NK cells cultured with rhIL-2 and with rhIL-15(P = 0.001). The expressions of NKG2D on edited Allo-NK cells cultured with rhIL-15 recovered to the same level as that of non-edited Allo-NK cells(P = 0.743). It showed that high dose rhIL-2 and rhIL-15 up-regulated the expressions of NKG2D. The efficacy of rhIL-15 was stronger than that of rhIL-2.The cytolytic effects of non-edited Allo-NK, edited Allo-NK cells, edited Allo-NK cells cultured with rhIL-2, and edited Allo-NK cells cultured with rhIL-15 on non-edited CNE2 cells were (35.90±3.27)%, (4.70±2.30)%, (31.70±3.56)% and (40.18±2.94)%, respectively. The differences of cytotoxicity were no significant between edited Allo-NK cells cultured with rhIL-2 or edited Allo-NK cells cultured with rhIL-15 and non-edited Allo-NK cells (P = 0.131, P = 0.124). The differences of cytotoxicity between edited Allo-NK cells cultured with rhIL-2 and edited Allo-NK cells cultured with rhIL-15 were significant (P = 0.009). It showed that high dose rhIL-2 and rhIL-15 recovered the cytotoxicity of edited Allo-NK cells and the efficacy of rhIL-15 was stronger than that of rhIL-2.The cytotoxicity of non-edited Allo-NK cells, edited Allo-NK cells cultured with rhIL-2, edited Allo-NK cells cultured with rhIL-15 was significantly different comparing with edited Allo-NK cells against edited CNE2 cells [(11.41±0.82)%, (10.43±1.21)%, (12.10±1.93)%, vs (0.49±0.05)%, respectively, P = 0.000 , P = 0.000 , P = 0.000]. The differences of cytotoxicity were no significant among groups of non-edited Allo-NK cells, edited Allo-NK cells cultured with rhIL-2, and edited Allo-NK cells cultured with rhIL-15 (P > 0.05). These findings suggested that high dose rhIL-2 and rhIL-15 recovered the cytotoxicity of edited Allo-NK cells against edited CNE2 cells.(2) Effects of DDP on expressions of NKG2D ligands and sensitivity of edited or non-edited CNE2 cells to Allo-NK cells cytolytic effectsThe expressions of MICA/MICB in non-edited CNE2 cells treated with 2.5μg/ml, 5μg/ml, and 10μg/ml DDP were (96.20±1.42)%, (96.93±1.33)% and (96.50±1.10)%, respectively, a comparison with that of the untreated group (87.47±1.80)% , showed significant increase in MICA/MICB expressions at all concentrations in the treated groups(P = 0.000, P =0.000, P =0.000). The expressions of MICA/MICB in edited CNE2 cells treated with 2.5μg/ml, 5μg/ml, and 10μg/ml DDP were (47.77±7.82)%, (58.07±1.92)% and (55.97±2.62)%, respectively, a comparison with data from the untreated group (35.20±2.00)%, showed distinctly increased MICA/MICB expressions at 5μg/ml and 10μg/ml of DDP treated groups(P < 0.05, P < 0.05). The differences of ULBP2 expressions among groups treated with 0μg/ml, 2.5μg/ml, 5μg/ml, and 10μg/ml DDP in edited and non-edited CNE2 cells were no significance (F= 3.231 , P = 0.082 ; F= 0.571 , P = 0.650, respectively).The results indicated that DDP up-regulated the expressions of MICA/MICB both on edited and non-edited CNE2 cells but no effects on ULBP2.At the E:T ratio of 20:1, the cytotoxicity of Allo-NK cells against non-edited CNE2 cells treated with 5μg/ml DDP was significantly increased comparing with that of control group [(47.71±1.53)% vs (38.11±1.41)%, P = 0.001]. The similar results were seen in edited CNE2 cells [(19.84±2.87)% vs (10.69±2.81)%, P = 0.017]. The data suggested that DDP enhanced the sensitivity of CNE2 cells to Allo-NK cells cytotoxicity.(3) Effects of 5-Fu on expressions of NKG2D ligands and sensitivity of edited or non-edited CNE2 cells to Allo-NK cells cytolytic effectsThe expressions of MICA/MICB in non-edited CNE2 cells treated with 5μM 5-Fu for 24 h had no distinction comparing with that of control [(85.70±1.05)% vs (87.47±1.80)%, t = 1.467, P = 0.216]. Nevertheless, the expressions of ULBP2 in non-edited CNE2 cells treated with 5μM 5-Fu were significant comparing with that of control [(68.00±0.50)% vs (47.40±0.66)%, t = 43.269, P = 0.000]. The expressions of MICA/MICB in edited CNE2 cells treated with 5uM 5-Fu for 24 h were no significant comparing with that of control [(34.93±1.63)% vs (35.20±2.00)% , t = 0.179, P = 0.866]. Nevertheless, the expressions of ULBP2 in edited CNE2 cells treated with 5μM 5-Fu were significant comparing with that of control [(62.43±1.72)% vs (45.83±1.33)%, t = 13.197, P = 0.000]. The results indicated that 5-Fu only up-regulated the expressions of ULBP2 both in edited and non-edited CNE2 cells, but exerted no effects on MICA/MICB expressions.At the E:T ratio of 20:1, the cytotoxicity of Allo-NK cells against non-edited CNE2 cells treated with 5μM 5-Fu was significantly increased comparing with that of control group[(51.25±3.71)% vs (38.11±1.41)%, t = 5.740, P = 0.005]. The similar results were seen in edited CNE2 cells [(24.38±4.28)% vs (10.69±2.81)%, t = 4.629, P = 0.01]. The data suggested that 5-Fu enhanced the sensitivity of non-edited and edited CNE2 cells to Allo-NK cells cytotoxicity.Conclusions 1 CNE2 cells expressed HLA-C1, C2 isotype molecules binding to Allo-NK cells inhibitory KIRs and MICA, MICB, ULBP2 binding to NKG2D activating receptors. The cytotoxicity of Allo-NK cells was regulated both by inhibitory and activating signal pathways, as a result, CNE2 cells were middle susceptible to Allo-NK cells cytolotic effects. K562 cells expressed MICA, MICB, ULBP1, ULBP2, and ULBP3 molecules binding to NKG2D receptors but no HLA-class I molecules binding to Allo-NK cells inhibitory KIR. The cytotoxicity of Allo-NK cells against K562 cells was regulated by activating signals but no inhibitory signals, resulted in high susceptibility to Allo-NK cells cytolotic effects.2 The existence of immunoediting between Allo-NK cells and CNE2 cells or K562 cells resulted in the decrease of Allo-NK cells cytotoxicity and susceptibility of CNE2 and K562 cells to Allo-NK cells cytolotic effects. The related mechanisms were the down-regulation of activating NKG2D receptors and the up-regulation of inhibitory KIRs of Allo-NK cells that resulted in one-shot killing of Allo-NK cells against tumor cells. Down-expressions of NKG2D ligands on CNE2 cells or K562 cells induced the weakened triggering and resistance to Allo-NK cells cytolytic effects. The molecular basis of immunoediting was the reciprocal reaction of HLA-KIR and NKG2DL-NKG2D signal pathways and the specificity of ligand-receptor binding.3 High dose rhIL-2 and rhIL-15 up-regulated the expressions of NKG2D in edited Allo-NK cells, and consequently recovered their cytotoxicity. DDP and 5-Fu up-regulated the expressions of MICA/MICB and ULBP2, respectively, in edited and non-edited CNE2 cells and accordingly enhanced triggering to Allo-NK cells cytotoxicity and sensitivity of CNE2 cells to Allo-NK cells cytolytic effects.4 The biologic behavior of edited Allo-NK cells and edited CNE2 cells could be changed by regulating inhibitory and activating signal pathways. Innovation of the current studyOur findings firstly unveiled that the existence of reciprocal-immunoediting between Allo-NK cells and CNE2 cells involved the interaction between HLA-KIR and NKG2DL-NKG2D signal pathways and specificity of ligand-receptor engagement, furthermore confirmed that regulating these two signal pathways could boost edited Allo-NK cells function and enhance sensitivity of edited CNE2 cells to Allo-NK cells cytotoxicity.Significance of the researchOur study provided a new insight into the relationship between Allo-NK cells and cancer cells, moreover, presented theoretical and experimental basis for establishment of a new potential genetic-based individual adoptive immuno-chemotherapeutic approach.
|
PDF Full Text Request